Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.
Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.
Müller
G
G
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Storz
P
P
Bourteele
S
S
Döppler
H
H
Pfizenmaier
K
K
Mischak
H
H
Philipp
A
A
Kaiser
C
C
Kolch
W
W
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D019556:COS Cells; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008934:Mitogens / Q000494:pharmacology / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*; D018631:ras Proteins / Q000378:metabolism
D002250:Carbon Radioisotopes; D002518:Ceramides; D008934:Mitogens; D014409:Tumor Necrosis Factor-alpha; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins